The long-term objectives of this application are to characterize the molecular mechanisms involved in the regulation of the intestinal absorption of vitamin B2 (riboflavin, RF), and those involved in intracellular trafficking and membrane targeting of the involved membrane transporter. We also aim at examining the effect of chronic alcohol use on intestinal RF absorption and determining the cellular and molecular mechanisms involved. RF is involved in critical cellular metabolic reactions, and thus, is essential for normal human health and well-being. Its deficiency, which occurs in a variety of conditions like chronic alcoholism, leads to serious clinical abnormalities that include degenerative changes in the nervous system, anemia, growth retardation, and skin lesions. Humans (and other mammals) cannot synthesize RF, and thus, must obtain the vitamin from exogenous sources via intestinal absorption. Thus, the human intestine plays a central role in maintaining and regulating normal RF body homeostasis. For these reasons understanding the physiology/pathophysiology as well as cell/molecular biology of the intestinal RF absorption process is of significant importance and is the aim of this proposal. We have been investigating the physiology of intestinal RF transport for over two decades, but more is needed at the molecular level to fully understand the process. With the recent molecular identification of mammalian RF transporters, we are now in an excellent position to carry out such investigations. Thus, in new preliminary studies we have shown that the up-regulation in intestinal RF uptake in RF deficiency shown by us previously is mediated (at least in part) via a transcriptional regulatory mechanism(s) affecting hRFT-2. We also showed that the intestinal RF uptake process undergoes differentiation - dependent regulation and that this regulation again appears to be mediated (at least in part) via a transcriptional mechanism(s). Using confocal imaging of living human intestinal epithelia cells and Western blot analysis of native human intestinal brush border and basolateral membrane preparations, our preliminary studies further showed exclusive expression of the human RF transporter -2 (hRFT-2; the most relevant intestinal RF transporter) at the apical membrane domain of polarized human intestinal epithelial cells. Furthermore, distinct trafficking vesicles appear to be involved in intracellular movement of hRFT2 in human intestinal epithelial cells. Finally, we showed for the first time that chronic alcohol feeding significantly inhibit RF transport across the jejunal BBM which is associated with a significant decrease in the level of expression of RFT-2. Our Specific Objectives in this proposal are: 1) To continue our investigations into the molecular mechanisms involved in the adaptive up-regulation in intestinal RF uptake in RF deficiency and during cell differentiation; 2) To study the mechanism(s) involved in the targeting of the hRFT-2 protein to the apical membrane domain of polarized human intestinal epithelial cells, and to determine the factor(s) involved in its intracellular trafficking; and 3) to extend our basic physiological/nutritional investigations on the intestinal RF uptake process into a clinically-relevant area and will examine the effect of chronic alcohol consumption on cell and molecular parameters of intestinal RF absorption process. Collectively, results of these studies should provide novel and valuable information regarding the physiology/pathophysiology and cell/molecular biology of intestinal RF uptake and of the factors that interfere with the process. This should ultimately assist us in the designing of effective strategies to optimize RF body homeostasis, especially in conditions of RF deficiency and sub- optimal levels. 1